Optical system for high resolution thermal melt detection

ABSTRACT

This invention relates to systems and methods for imaging sample materials within a microfluidic device during an assay reaction process. In accordance with certain aspects of the invention, images are formed with a pixel array and a region of interest (“ROI”) is defined within the pixel array. Image values, such as fluorescent intensity, can be computed as averages of individual pixel values within the ROI. Where the ROI is subject to non-uniform conditions, such as non-uniform heating, the ROI can be divided into sub-ROIs which are sufficiently small that the condition is uniform within the sub-ROI.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/222,487 filed Aug. 31, 2011, which claims the benefit of priority toU.S. Provisional Application No. 61/378,471, filed Aug. 31, 2010, thedisclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND

1. Field of the Invention

This invention relates to systems and methods for imaging samplematerials within a microfluidic device during an assay reaction process.

2. Discussion of the Background

The detection of nucleic acids is central to medicine, forensic science,industrial processing, crop and animal breeding, and many other fields.The ability to detect disease conditions (e.g., cancer), infectiousorganisms (e.g., HIV), genetic lineage, genetic markers, and the like,is ubiquitous technology for disease diagnosis and prognosis, markerassisted selection, correct identification of crime scene features, theability to propagate industrial organisms and many other techniques.Determination of the integrity of a nucleic acid of interest can berelevant to the pathology of an infection or cancer. One of the mostpowerful and basic technologies to detect small quantities of nucleicacids is to replicate some or all of a nucleic acid sequence many times,and then analyze the amplification products. Polymerase chain reaction(PCR) is a well-known technique for amplifying DNA.

With PCR, one can quickly produce millions of copies of DNA startingfrom a single template DNA molecule. PCR includes a three phasetemperature cycle of denaturation of the DNA into single strands,annealing of primers to the denatured strands, and extension of theprimers by a thermostable DNA polymerase enzyme. This cycle is repeateda number of times so that at the end of the process there are enoughcopies to be detected and analyzed. For general details concerning PCR,see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rdEd.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(2000); Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2005) and PCR Protocols A Guide to Methods and Applications, M. A. Inniset al., eds., Academic Press Inc. San Diego, Calif. (1990).

In some applications, it is important to monitor the accumulation of DNAproducts as the amplification process progresses. Real-time PCR refersto a growing set of techniques in which one measures the buildup ofamplified DNA products as the reaction progresses, typically once perPCR cycle. Monitoring the amplification process over time allows one todetermine the efficiency of the process, as well as estimate the initialconcentration of DNA template molecules. For general details concerningreal-time PCR see Real-Time PCR: An Essential Guide, K. Edwards et al.,eds., Horizon Bioscience, Norwich, U.K. (2004).

More recently, a number of high throughput approaches to performing PCRand other amplification reactions have been developed, e.g., involvingamplification reactions in microfluidic devices, as well as methods fordetecting and analyzing amplified nucleic acids in or on the devices.Thermal cycling of the sample for amplification is usually accomplishedin one of two methods. In the first method, the sample solution isloaded into the device and the temperature is cycled in time, much likea conventional PCR instrument. In the second method, the sample solutionis pumped continuously through spatially varying temperature zones. See,for example, Lagally et al. (Anal Chem 73:565-570 (2001)), Kopp et al.(Science 280:1046-1048 (1998)), Park et al. (Anal Chem 75:6029-6033(2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No.6,960,437) and Knapp et al. (U.S. Patent Application Publication No.2005/0042639).

Further examples of systems, methods, and apparatus for high throughputapproaches to performing PCR and other amplification reactions aredescribed in the following publications that are related to the subjectmatter of the present disclosure.

U.S. Patent Application Publication No. 2008/0176230 to Owen et al.entitled “Systems and methods for real-time PCR” (the '230publication”), the disclosure of which is hereby incorporated byreference, describes systems and methods for the real-time amplificationand analysis of a sample of DNA within a micro-channel.

U.S. Patent No. 7,629,124 to Hasson et al. entitled “Real-time PCR inmicro-channels” (the '124 patent”) the disclosure of which is herebyincorporated by reference, describes systems and methods for performingreal time PCR in micro-channels by continuously moving boluses of testsolution separated by carrier fluid through the micro-channels andperforming a process, such as PCR, on each bolus and measuring signals,such as fluorescent signals, at different locations along a definedsection of the channel.

U.S. Patent No. 7,593,560 to Hasson et al. entitled “Systems and methodsfor monitoring the amplification and dissociation behavior of DNAmolecules” (the '560 patent”), the disclosure of which is herebyincorporated by reference, describes the use of sensors for monitoringreactions within microfluidic channels. The sensor has a defined pixelarray for collecting image data, and image data from a select window ofpixels (a sub-set of the entire array), which encompasses a portion ofinterest of a micro-channel, is processed and stored for each of themicro-channels.

Once there are a sufficient number of copies of the original DNAmolecule, the DNA can be characterized. One method of characterizing theDNA is to examine the DNA's dissociation behavior as the DNA transitionsfrom double stranded DNA (dsDNA) to single stranded DNA (ssDNA) withincreasing temperature. The process of causing DNA to transition fromdsDNA to ssDNA is sometimes referred to as a “high-resolutiontemperature (thermal) melt (HRTm)” process, or simply a “high-resolutionmelt” process.

To monitor a PCR process and/or a melting process (quantitatively and/orqualitatively), an imaging system may be employed to measure anoptically detectable characteristic, such as fluorescence, of a dye thatis incorporated into the sample material and that varies in a detectablemanner as the number of copies of the original DNA molecule increasesand/or as the DNA transitions from double stranded DNA (dsDNA) to singlestranded DNA (ssDNA) with increasing temperature. The accuracy andreliability of nucleic acid assays depends, to a large extent, on theaccuracy and precision of such imaging systems. Moreover, the costs ofsuch imaging systems are a significant portion of the cost of an overallinstrument for performing nucleic acid assays.

Thus, there is a continuing need for improvements in accuracy,precision, and cost effectiveness of imaging systems for monitoringnucleic acid diagnostic assays and other biological processes.

SUMMARY

Using a 2 dimensional CMOS or CCD sensor to image a fluorescence sourceis known in biological studies. Usually a microscope lens is used toimage the fluorescence source into scientific CMOS/CCD sensor. Inaccordance with aspects of the present invention, improved imagingand/or lower cost imaging systems are achieved by using a digital singlelens reflex (“DSLR”) camera as the imaging device in combination withLED excitation sources of a prescribed configuration and arrangementdescribed herein. Such an imaging system has many advantages, including:

(1) The large CMOS sensor of the DSLR camera allows both flow trackingand thermal melt measurements using the same camera.

(2) The 8-bit JPEG format of the DSLR camera saves data transferbandwidth and hard drive spaces compared with 14-bit RAW data. Bit depthcan be restored by averaging many pixels.

(3) A sensor with large pixel density, such of the DSLR camera, permitsreactions in the microfluidic channel to be observed. Information suchas bubble formation could be obtained to have better control of the PCRand thermal melt process.

(4) Due to large CMOS sensor size of the DSLR camera, referencefluorescence materials could be used to correct for and removefluctuations from the light source and the heater.

Thus, aspects of the invention are embodied in an imaging systemconfigured to generate images of a reaction within a microchannel of amicrofluidic device. In one embodiment, the imaging system comprises asensor element configured to generate a storable image of at least aportion of a microchannel and a plurality of illumination elementsdisposed with respect to the sensor element and configured to illuminatea portion of the microfluidic chip to be imaged by the sensor element.At least one of the illumination elements comprises an illuminationassembly comprising an LED, a mask disposed in front of the LED andhaving an opening formed therein so as to control an area illuminated bythe illumination assembly, a filter along an optic path of theillumination assembly for controlling the spectral content of lightemitted by the illumination assembly, and a lens for imaging an areawith light emitted by the illumination assembly. The LED, the mask, thefilter, and the lens are aligned along an optic axis of the illuminationassembly.

In one embodiment, at least two of the illumination elements areconfigured to illuminate different portions of the microfluidic chip.

In another embodiment, the sensor element comprises a digital singlelens reflex camera.

In another embodiment, each of the illumination elements comprises anLED.

In another embodiment, the imaging system comprises four illuminationelements disposed at 90-degree angular increments about the sensorelement.

In another embodiment, the microchannel comprises a first zone and asecond zone, wherein a first one of the LEDs is positioned and orientedto illuminate the second zone, a second and a third of the LEDs arespaced 180-degrees from each other and are disposed on opposed sides ofthe sensor and are positioned and oriented to illuminate the first zone,and wherein a fourth one of the LEDs is positioned and oriented toilluminate both the first zone and the second zone.

In another embodiment, the sensor element comprises a CMOS sensor.

In another embodiment, the CMOS sensor has a pixel array of up to5616×3744 pixels or higher.

In another embodiment, the sensor element includes a pixel array, andthe system further comprises logic elements configured to detect animage of only a portion of the pixels of the pixel array.

In another embodiment, the sensor element is configured to generatemultiple images at a frequency of up to 30 Hz or higher.

In another embodiment, the sensor element is configured to generate animage having an 8-bit JPEG format.

In another embodiment, the imaging system further comprises referencefluorescence material positioned to be imaged by the sensor elementalong with at least a portion of a microchannel.

In another embodiment, the imaging system further comprises at least oneextension tube between the sensor and the microchannel. In otherembodiments, lens combinations may be used in place of extension tubes.

In another embodiment, the imaging system further comprises an emissionfilter positioned between the sensor and the microchannel and isconfigured to allow light signals of only a selected wavelength to reachthe sensor.

In another embodiment, each of the plurality of illumination elements isconfigured to illuminate a portion of the microfluidic chip at aprescribed wavelength.

Further aspects of the invention are embodied in a system for performinga nucleic acid diagnostic assay on a sample material. In one embodiment,the system comprises microfluidic means including micro-channels fortransporting sample material and for enabling an assay process to beperformed on sample material within one or more portions of themicro-channels. The assay process includes at least one of PCRamplification and/or thermal melt analysis. The system further includesmeans in operative cooperation with the microfluidic means forintroducing sample material into the micro-channels of the microfluidicmeans; means in operative cooperation with the microfluidic means formoving sample material through each micro-channel of the microfluidicmeans, thermal means for heating and/or cooling one or more portions ofthe microfluidic means to one or more selected temperatures, and imagingmeans for imaging sample material within one or more portions of eachmicro-channel, including means for storing data related to imagescreated by the imaging means.

In another embodiment, the system further comprises processing means forprocessing data related to images created by the imaging means and forgenerating data relating to results of at least one of the PCRamplification and the thermal melt analysis.

In another embodiment, the system further comprises control means forcontrolling operation of the means for introducing sample material, themeans for moving sample material, the thermal means, the imaging means,and the processing means.

In another embodiment, the imaging means is configured for detectingfluorescent emissions of prescribed wavelengths from sample materialwithin the micro-channels and comprises means for directing anexcitation signal of a prescribed excitation wavelength at a portion ofthe micro-channel and means for capturing an image of fluorescentemission of a prescribed emission wavelength from the sample materialwithin the portion of the micro-channel.

Further aspects of the invention are embodied in a computer-implementedmethod for analyzing thermal melt data from an image of a reactionwithin a microchannel of a microfluidic device. In one embodiment, themethod comprises the steps of illuminating at least a portion of themicrochannel, generating, with a pixel array sensor, an image offluorescence emitted by material within the illuminated portion of themicrochannel, wherein each pixel of the image has a JPEG value, defininga region of interest (“ROI”) comprising a portion of the pixels of thepixel array, and calculating the intensity of fluorescence emitted bymaterial within the microchannel by averaging the JPEG values of all thepixels in the ROI.

In another embodiment, each pixel has two sub-pixels of a first colorand one sub-pixel of a second color, and the JPEG value of each pixel iscomputed as (2×first color sub-pixel+1×second color sub-pixel)/3.

In another embodiment, the method further comprises dividing at least aportion of the ROI into one or more sub-ROIs and calculatingfluorescence intensity within each sub-ROI by averaging JPEG values ofall the pixels in the sub-ROI.

In another embodiment, the generating step is performed with a CMOSsensor.

In another embodiment, the CMOS sensor comprises a digital single lensreflex camera.

In another embodiment, the method further comprises repeating thegenerating and calculating steps one or more times over a period of timeand monitoring changes in the calculated fluorescence intensity over aperiod of time.

In another embodiment, the method further comprises monitoring a fluidflow within the microchannel by generating images of fluorescenceemitted by material within the illuminated portion of the microchannelat two different times, detecting displacement of a feature of the imagewithin from one image to the next, and computing a time lapse from oneimage to the next.

In another embodiment, the method further comprises illuminating areference fluorescence material positioned adjacent the microchannel,generating, with the pixel array sensor, an image of fluorescenceemitted by the reference fluorescence material, and defining at leasttwo ROIs, wherein one ROI encompasses the portion of the microchanneland a second ROI encompasses a portion of the reference fluorescencematerial.

In another embodiment, the method further comprises calculating theintensity of fluorescence emitted by the reference fluorescence materialby averaging the JPEG values of all the pixels in the ROI encompassingthe reference fluorescence material and adjusting the intensity offluorescence emitted by material within the microchannel based on theintensity of fluorescence emitted by the reference fluorescencematerial.

Further aspects of the invention are embodied in a computer-implementedmethod for analyzing thermal melt data. In one embodiment, the methodcomprises recording thermal melt image data as a function of time withan imaging system by saving JPEG images generated by the imaging systemwith time stamps, recording temperature data as a function of time witha temperature control system, and synchronizing the thermal melt datawith the temperature data by sending a static record signal to theimaging system to cause the imaging system to record static image databefore thermal melt is started, recording the time of the static imagedata, and synchronizing the temperature data to the time of the staticimage data.

In another embodiment, the method comprises sending the static imagesignal to the imaging system at the same time an initial heater signalis sent to the temperature control system, so that the thermal meltimage data and heaters controlled by the temperature control system willhave the same start time, and then synchronizing the temperature dataand the thermal melt image data based on a relationship of the time ofthe static image data to the time stamps of the JPEG images of thethermal melt image data and recorded time of the temperature data.

These and other features, aspects, and advantages of the presentinvention will become apparent to those skilled in the art afterconsidering the following detailed description, appended claims andaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIG. 1 is a block diagram illustrating a system in which an imagingsystem incorporating aspects of the invention can be incorporated.

FIG. 2 is a perspective view of an imaging system including a sensor andLED layout according to an embodiment of the invention.

FIG. 3 is a side view of an imaging system including a sensor and LEDlayout according to an embodiment of the invention.

FIG. 4 is a schematic view of an LED assembly of the imaging systemaccording to an embodiment of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Systems for nucleic acid analyses using microfluidic chips with one ormore micro-channels, such as the system described in the aforementioned'230 publication, the '560 patent, and the real-time PCR architecturedescribed in the aforementioned '124 patent, include an image sensor (oroptical imaging system) for detecting optically-detectablecharacteristics of a sample flowing through a micro-channel, such asamplification products and flow rate of the test solution. Test solutionflowing through each micro-channel may be in the form of discreteboluses of sample solution separated by carrier fluid, as described inthe '124 patent.

FIG. 1 is a block diagram illustrating a system 100 for rapid serialprocessing of multiple nucleic acid assays that can be configured toembody various aspects of the invention. System 100 may include amicrofluidic device 102. Microfluidic device 102 may include one or moremicrofluidic channels 104. In the examples shown, device 102 includestwo microfluidic channels, channel 104 a and channel 104 b. Althoughonly two channels are shown in the exemplary embodiment, it iscontemplated that device 102 may have fewer than two or more than twochannels. For example, in some embodiments, device 102 includes eightchannels 104.

In one embodiment, device 102 may include two DNA processing zones, aDNA amplification zone 131 (a.k.a., PCR zone 131) and a DNA thermalmelting zone 132. A DNA sample traveling through the PCR zone 131 mayundergo PCR, and a DNA sample passing through thermal melting zone 132may undergo high resolution thermal melting. As illustrated in FIG. 1,PCR zone 131 includes a first portion of channels 104 and thermalmelting zone 132 includes a second portion of channels 104, which isdown stream from the first portion.

Device 102 may also include a sipper 108. Sipper 108 may be in the formof a hollow tube. Sipper 108 has a proximal end that is connected to aninlet 109 which inlet couples the proximal end of sipper 108 to channels104. As an alternative to, or in addition to, the sipper 108, the systemmay include a liquid handling system comprising at least one roboticpipettor for aspirating, mixing, and dispensing reagent and/or samplemixtures to the microfluidic cartridge 102.

Device 102 may also include a common reagent well 106 which is connectedto inlet 109. Device 102 may also include a locus specific reagent well105 for each channel 104. For example, in the embodiment shown, device102 includes a locus specific reagent well 105 a, which is connected tochannel 104 a, and may include a locus specific reagent well 105 b whichis connected to channel 104 b. Device 102 may also include a waste well110 for each channel 104.

The solution that is stored in the common reagent well 106 may containdNTPs, polymerase enzymes, salts, buffers, surface-passivating reagents,one or more non-specific fluorescent DNA detecting molecules, a fluidmarker and the like. The solution that is stored in a locus specificreagent well 105 may contain PCR primers, a sequence-specificfluorescent DNA probe or marker, salts, buffers, surface-passivatingreagents and the like.

In order to introduce a sample solution into the channels 104, system100 may include a well plate 196 that includes a plurality of wells 198,at least some of which contain a sample solution (e.g., a solutioncontaining a nucleic acid sample). In the embodiment shown, well plate196 is connected to a positioning system 194 which is connected to amain controller 130.

In one non-limiting embodiment, main controller 130 may be implementedusing a PXI-8105 controller which is available from National InstrumentsCorporation of Austin, Tex. In one non-limiting embodiment, positioningsystem 194 may include a positioner (e.g., the MX80 positioner availablefrom Parker Hannifin Corporation of PA (“Parker”)) for positioning wellplate 196, a stepping drive (e.g., the E-AC Microstepping Driveavailable from Parker) for driving the positioner, and a controller(e.g., the 6K4 controller available from Parker) for controlling thestepping drive.

In one embodiment, to introduce a sample solution into the channels 104,the positioning system 194 is controlled to move well plate 196 suchthat the distal end of sipper 108 is submerged in the sample solutionstored in one of the wells 198. FIG. 1 shows the distal end of 108 beingsubmerged within the sample solution stored in well 198 n.

In order to force the sample solution to move up the sipper and into thechannels 104, a vacuum manifold 112 and pump 114 may be employed. Thevacuum manifold 112 may be operably connected to a portion of device 102and pump 114 may be operably connected to manifold 112. When pump 114 isactivated, pump 114 creates a pressure differential (e.g., pump 114 maydraw air out of a waste well 110), and this pressure differential causesthe sample solution stored in well 198 n to flow up sipper 108 andthrough inlet channel 109 into channels 104. Additionally, this causesthe reagents in wells 106 and 105 to flow into a channel. Accordingly,pump 114 functions to force a sample solution and real-time PCR reagentsto flow through channels 104. As illustrated in FIG. 1, melt zone 132 islocated downstream from PCR zone 131. Thus, a sample solution will flowfirst through the PCR zone and then through the melting zone.

Referring back to well plate 196, well plate 196 may include a buffersolution well 198 a. In one embodiment, buffer solution well 198 a holdsa buffer solution 197. Buffer solution 197 may comprise a conventionalPCR buffer, such as a conventional real-time (RT) PCR buffer.Conventional PCR buffers are available from a number of suppliers,including, for example: Bio-Rad Laboratories, Inc., Applied Biosystems,Roche Diagnostics, and others.

In order to replenish buffer solution well 198 a with the buffersolution 197, system 100 may include a buffer solution storage container190 and a pump 192 for pumping the buffer solution 197 from container190 into well 198 a. Additionally, pump 192 may be configured to notonly add solution 197 to well 198 a, but also remove solution 197 fromwell 198 a, thereby re-circulating the solution 197.

In one configuration, as described in the '124 patent, the systemincludes a test solution reservoir, which as described above, may be areservoir containing multiple test solutions, such as a multi-well,microtiter plate 196 in which each well contains different testsolutions, e.g., test samples. The system further includes a carrierfluid reservoir. In one embodiment, the test solution is substantiallythe same as the carrier fluid, except that the test solution comprisesall the necessary real-time PCR reagents. The real-time PCR reagentmixture may include PCR primers, dNTPs, polymerase enzymes, salts,buffers, surface-passivating agents, and the like. In addition, thereal-time PCR mixture may include a non-specific fluorescent DNAdetecting molecule, a sequence-specific fluorescent DNA probe or amarker. In an additional embodiment, the carrier fluid is an immisciblefluid (such as an oil, a fluorinated liquid, or any other nonaqueous orhydrophobic solvent). The purpose of the carrier fluid is to detertransfer of material from one test bolus to another. Another purpose ofthe carrier fluid is to provide a distinguishable transition betweenboluses that may be used to track the fluid flow in the channel. In oneembodiment, the carrier fluid may include a marker.

In one embodiment, the test solution and carrier fluid are introducedinto a microchannels 104 a, 104 b through a switch (not shown) undercontrol of the main controller 130 such that the carrier fluid and thetest solution are sequentially alternately introduced into microchannels104 a, 104 b to form discrete boluses of test solution separated fromone another by carrier fluid. The volume of the test solution andcarrier fluid that is introduced into microchannels 104 a, 104 b isselected such that there is minimal blending between them duringmovement through microchannels 104 a, 104 b.

A multitude of reactions in series (or sequential reactions) can thus becarried out in each of the microchannels 104 a, 104 b as a result of thecontinuous movement of boluses of different test solutions throughmicrochannels 104 a, 104 b, each separated by the carrier fluid. Theflow rate of the carrier fluid and test solution boluses throughmicrochannels 104 a, 104 b is controlled by pump 114 under control ofmain controller 130 in order to regulate the flow rate of the testsolution boluses and the carrier fluid in microchannels 104 a, 104 b.The flow rate may be regulated such that a desired number of PCR cyclesare performed as the test solution boluses passes through PCR zone 131of the microchannels 104 a, 104 b.

In order to achieve PCR for a DNA sample flowing through the PCR zone131, the temperature of the sample must be cycled, as is well known inthe art. Accordingly, in some embodiments, system 100 includes atemperature control system 120. The temperature control system 120 mayinclude a temperature sensor, a heater/cooler, and a temperaturecontroller. In some embodiments, a temperature control system 120 isinterfaced with main controller 130 so that main controller 130 cancontrol the temperature of the samples flowing through the PCR zone andthe melting zone. Although a single temperature control system 120 isshown for the entire microchip 102, the temperature control system 120may comprise separate temperature control sub-systems—each comprising,for example, a temperature sensor, a heater/cooler, and a temperaturecontroller—for the PCR zone 131 and the melt zone 132.

Main controller 130 may be connected to a display device for displayinga graphical user interface. Main controller 130 may also be connected touser input devices 134, which allow a user to input data and commandsinto main controller 130.

To monitor the PCR process and the melting process that occur in PCRzone 131 and melt zone 132, respectively, system 100 may include animaging system 118. Imaging system 118 may include an excitation source,an image capturing device, a controller, and an image storage unit.According to an embodiment of the invention, the imaging system 118comprises a CMOS sensor that, in one embodiment, may comprise part of anoff-the-shelf digital single-lens reflex (DSLR) camera, which has alarge format CMOS sensor (such as, for example, 24 mm×36 mm). The fieldof view is much larger than prior systems that employ microscope lensesand allows imaging of the entire microfluidic chip. The imaging system118 could be used for both flow tracking of the microfluidic system andthermal melt signal detection.

Methods for flow tracking, i.e., measuring flow rate through the microchannels 104 a, 104 b, are described in the '124 patent. For example, inone embodiment, the average flow rate is measured by comparingsequential images of the reaction-dependent fluorescent signal from thechannel. In a second embodiment, the average flow rate is measured bycomparing sequential images of a reaction-independent flow marker fromthe channel. More specifically, the system acquires one or more imagesof the contents of the channel. These image data and the time ofacquisition are stored to a database for subsequent analysis. A featureof two or more sequential images—e.g., reaction-dependent fluorescentsignal from the channel or reaction-independent flow marker from thechannel—may be compared to determine how far the fluid has moved alongthe channel from one image to a subsequent image. Dividing the averagedisplacement by the elapsed time gives an average flow speed. In anotherembodiment, scattered light from the reaction-independent flow marker isresolvable from the reaction-dependent fluorescence by wavelengthspectrum. In an alternative embodiment, scattered light from thereaction-independent flow marker is resolvable from thereaction-dependent fluorescence on the basis of fluorescence lifetime.In another embodiment, the reaction-independent flow marker is furtherused to determine the flow dispersion of the test bolus. In a furtherembodiment, the image of reaction-dependent fluorescence is captured atleast once per PCR cycle. In one embodiment, the image ofreaction-dependent fluorescence is captured sequentially by scanning alength of the channel on a time scale shorter than the duration of onePCR cycle. In an alternative embodiment, the image of reaction-dependentfluorescence is captured by acquiring signals from multiple points alongthe channel simultaneously. In another embodiment, the flow ratemeasurements are part of a feedback loop for regulating the flow rate.In a further embodiment, the flow rate is measured through detecting asample bolus entrance into and exit from a defined section of thechannel.

In some embodiments, the determined flow rate may be used as an input tocontrol the pump 114, via the main controller 130, in a feedback flowcontrol loop.

More specifically, in an embodiment of the invention, a digitalsingle-lens reflex (DSLR) camera is employed for high resolution thermalmelt and flow tracking in microfluidic chips. In a non-limiting example,the DSLR camera can be a Canon EOS 5D Mark II. The DSLR camera is alsosuitable to acquire real-time PCR information (as described, forexample, in the '124 patent) when applicable. Usually, digital camerassave pictures in RAW or JPEG format. To achieve a fast frame rate, JPEGformat is usually adopted. In one non-limiting embodiment, the DSLRcamera has a CMOS sensor of 24 mm×36 mm, with a pixel array of5616×3744, or higher, arranged in a Bayer pattern (2 Green, 1 Blue, 1Red sub-pixel for each pixel).

To record fluorescence change over time during DNA thermal melt, a highframe rate is preferred. In one embodiment, the detector can be adigital color camera that is capable of recording data at video framesrates, such as, for example, 20-30 frames per second. The live viewfunction of a suitable DSLR camera, such as, for example, the Canon EOS5D Mark II, provides a frame rate of 30 fps. Of course, a suitable DSLRcamera with a higher or lower frame rate may be used as well in certainembodiments. In a non-limiting example, files from the live viewfunction are saved in JPEG format with two choices of resolutions:

a. full sensor images with sub-sampled pixels, image size 1024×680 (1×zoom); and

b. a crop of the sensor with full pixel resolution, image size 1120×752(5× zoom).

In one embodiment, the 1× zoom is used for flow tracking, since itimages a relatively large area (at a reduced pixel density), which mayinclude portions of the microfluidic chip where PCR and thermal meltoccur and part of an interface chip coupled to the microfluidic chip forliquid handling. In one embodiment, the 5× zoom is used for thermal meltdetection where increased resolution (detail) of a small portion of themicrofluidic chip is required. In a preferred embodiment, all the pixelsin the imaged area are preserved so better signal to noise ratio (SNR)can be achieved. In other embodiments, a different zoom setting could beused for either flow tracking or thermal melt detection.

JPEG image format offers 8-bit resolution, i.e., 256 digital levels foreach pixel. In some embodiments, more bit depth could be achieved byaveraging many pixels in the region of interest (“ROI”) to achievegreater than 8-bit resolution.

As described in the '560 patent, the pixel array of the sensor may bedivided into zones of interest corresponding to particular ROI's. Thatis, the image sensor has the ability to read out or “window” apredefined portion of the pixel array (this is known as “windowing”).For thermal melt imaging, the melting zone 132 of the micro-channel 104a, 104 b is uniformly illuminated with a high power LED and is imagedinto the CMOS sensor with ˜1:1 ratio. In the melting zone 132 wherethermal melt is measured, the fluorescence intensity is calculated byaveraging the JPEG values of all the pixels in the ROI. In oneembodiment, fluorescence intensity is calculated by averaging JPEGvalues of >2000 pixels. By averaging pixel JPEG values (8-bit) of an ROIwith numerous pixels, better than 11 bit resolution could be achieved.

Another benefit from averaging the pixels in the ROI is to improvesignal-to-noise ratio (“SNR”).

Fluorescence from LC Green (DNA binding dye used for high resolutionmelting) is nearly equally sensed by green and blue pixels of the CMOSsensor. Thus, the average of the green and blue pixels is used torepresent the fluorescence intensity on that specific pixel of thesensor. Because of the Bayer filter in front of the sensor in accordancewith one embodiment, in each pixel there are 2 green pixels and 1 bluepixel. Accordingly:

Pixel fluorescence intensity=(2*Green+1*Blue)/3.

In reality, the weight of Green and Blue may deviate from the 2:1 ratiodue to other camera settings. One such setting that can affect the Greento Blue ratio is white balance. White balance manipulates green, blue,and red pixel JPEG values to adapt the image to different illuminationconditions. Using SNR as a metric, optimized condition of white balancesettings and green, blue ratio could be calculated or measured.

An imaging system 10, according to an embodiment of the invention, isshown in FIGS. 2 and 3. The imaging system 10 includes a CMOS sensor 36,which, in one embodiment, is housed within the body 14 of a camera 12,for example, a DSLR camera, such as the Canon EOS 5D Mark II, and a lensbarrel 16 containing one or more lenses 32. In embodiments of theinvention, the imaging system 10 may be used for thermal meltmeasurement and flow tracking In one embodiment, the imaging system 10is configured to generate and record multiple images per second for flowtracking and thermal melt analysis. For example, the Canon EOS 5D MarkII includes a live view mode that is used to generate and record JPEGimages at ˜1 Hz for flow tracking purposes while boluses flow inmicrofluidic channels. It could also be used for real-time PCR purposes.In certain embodiments, for thermal melt data recording, images, such asJPEG files, are recorded at ˜30 Hz rate, with integration time ˜33 ms(i.e., the integration time interval is computed as 1/freq.). Otherframe rates and corresponding integration time intervals may beemployed, depending on system requirements and hardware capability, butframe rates are preferably not less than 10 Hz.

In one embodiment, an F/1.2 lens, which is commercially available, maybe used for greater photon collection. Lenses with smaller F/# havehigher light collection capability.

Extension tubes 15, 13 may be used to make the distance between lens 16and the microfluidic chip to be imaged as close as possible. Lightcollection is proportional to the square of distance between lens andfluorescence source. In one embodiment, the extension tubes have lengthsof 25 mm and 12 mm, for a total extension length of 37 mm, which canmake the distance between lens 16 and the microfluidic chip to be imagedas close as about 6 cm. Other extension tube lengths could be used, suchas, for example, 25 mm and 25 mm. In addition, more or less than twoextension tubes can be employed. For example, other embodiments mayinclude three extension tubes of 25 mm, 25 mm, and 12 mm or 25 mm, 25mm, and 25 mm. Longer extension tubes make the distance between lens andthe microfluidic chip smaller, which allows more light to enter the lensand which could also could make the image on the sensor larger. Thechoice of extension tubes may depend on specific mechanical design andfield of view considerations. As an alternative to the use of extensiontubes, lens combinations could be used to achieve the same goal ofmoving the lens closer to the sample material in the microfluidic chipand providing larger images.

The sensor may include a dual band emission filter 34, which may befitted in one of the extension tubes 15, 13 to allow only selectedwavelengths to reach the sensor. In one embodiment, the filter isconfigured to allow only fluorescence from LC Green and Alexa 647 (orTexas Red) tracking dye to reach the sensor. In one exemplaryembodiment, the emission filter is a dual bandpass filter with apass-band for the DNA binding dye LC Green Plus from Idaho Technologyand a pass-band for a red flow tracking dye such as AlexaFluor 647 fromLife Technologies. However, alternative filters may be substituted forappropriate alternative combinations of fluorescent dyes. Fluorescencefrom LC Green has a blue-green color that is sensed by both blue andgreen pixels of the CMOS sensor. However, because the camera's greenfilter will block blue photons, and the camera's blue filter will blockgreen photons, at least half of the photons are rejected by eitherfilter. In one embodiment, the filter has a shifted spectrum (e.g., tothe blue-green wavelength) so all the photons could pass the filter toreach the sensor pixels. The camera's red filter can be kept unchangedso the ‘dual color’ sensor can still do both flow tracking and thermalmelt detection with two different dyes.

In one embodiment, the imaging system 10 includes four LEDs 1, 2, 3, 4for illumination of the microfluidic chip. The LEDs are excitationsources that generate light at desired wavelengths to excite labels usedfor detecting amplification products during real-time PCR, dissociationbehavior during thermal melt analysis, and/or to detect markers that maybe present to monitor the flow rate of the test solution in microchannel104 a and/or 104 b. As explained above, PCR zone 131 of the chip is forPCR, and melting zone 132 is for thermal melt. An embodiment of thelayout of the sensor 12 and LEDs 1, 2, 3, 4, of the imaging device 10 isshown in FIGS. 2 and 3. FIG. 2 is an illustration of an imaging device10, including the sensor (e.g., camera) 12 and the LED layout. As shownin the illustrated embodiment, the LEDs 1, 2, 3, 4 are arranged adjacentto the lens(es) 16 (lens barrel) at 90° angular intervals around thelens 16. LEDs 2 and 3 are disposed opposite each other with respect tothe lens 16, and LEDs 1 and 4 are disposed opposite each other withrespect to the lens 16.

In an exemplary embodiment, LED 1 comprises an assembly with a slotaperture and an imaging lens and is positioned and oriented for meltingzone 132 (thermal melt) illumination. LED 2 and LED 3 are positioned andoriented for PCR zone 131 (PCR) heater calibration and can also be usedto monitor PCR process and melt in zone 131 if necessary. LED 4 ispositioned and oriented to excite flow tracking dye in the whole chip102. In one embodiment, a goal is to limit illumination of LED 4 to PCRzone 132, thus avoid photobleaching in melting zone 131 when doingthermal melt.

FIG. 4 is a schematic view of the melting zone 132 LED (LED 1) assemblyin accordance with one embodiment. As shown in FIG. 4, LED 1 is actuallyan assembly that includes an LED 30, a mask 28, a filter 26, and a lens24 aligned along an optic axis 22. Mask 28 includes a slot for limitingthe illumination from the LED 30 to thermal melt zone 132 of themicrofluidic chip 102. Filter 26 controls the spectral content ofexcitation light directed at the chip 102, and lens 24 images the lightband onto melting zone 132. Melting zone 132 is illuminated with theband shape light from the mask 28 of the LED assembly 1. With theband-shaped light, this LED (blue LED) illuminates only melting zone132, so as to avoid photo-bleaching of the dye in PCR zone 131. Theassembly may also include heat sinks and fans.

In one embodiment, two high-powered blue LEDs (2, 3) are placed at bothsides of PCR zone 131 to excite LC Green in zone 131 for heatercalibration. In an embodiment of a system for nucleic acid analysesusing a microfluidic chip with one or more micro-channels, the heatingelement may comprise metal wires or filaments disposed adjacent to themicro-channels, for example, as disclosed in U.S. Patent ApplicationPublication No. 2009-0248349 “Microfluidic Devices with IntegratedResistive Heater Electrodes Including Systems and Methods forControlling and Measuring the Temperature of Such Heater Electrodes” andU.S. Patent Application Publication No. 2011-0048547 “Microfluidicsystems and methods for thermal control,” the disclosures of which arehereby incorporated by reference in their entireties.

In one embodiment, LEDs 2 and 3 are disposed on opposite sides of themicrofluidic chip 102 to avoid shadow in PCR zone 131 due to the heaterelements. Additionally, this disposition of LEDs 2 and 3 creates moreuniform illumination across all channels. Heater calibration can beperformed by passing a substance with a known nucleic acid concentrationthrough a micro-channel 104 a, 104 b, illuminating melting zone 132 withLED 1 and generating thermal melt curves that are compared to expectedthermal melt curves for the known nucleic acid concentration. In someembodiments, LEDs 2 and 3 could also be used to monitor PCR and thermalmelt in zone 131.

In one embodiment, LED 4 (red LED) flood illuminates the whole chip 102,which can be used for flow tracking purposes

In preferred embodiments, all of the LEDs 1, 2, 3, 4 and the sensor 12are facing up, and the transparent side of the microfluidic chip 102faces down. This arrangement allows a liquid handling system (e.g.,robotic pipetter) to use the space above the chip 102 with minimalobstacles.

In preferred embodiments, LEDs 1, 2, 3, 4 are controlled by a controllerof the imaging system 118 and/or the main controller 130 executingcontrol algorithms written, in one non-limiting example, in Labview fromNational Instruments. Other control algorithms also can be used as wouldbe known by persons skilled in the art.

It is understood that the CMOS sensor of imaging system 10 need not be aDSLR camera. In embodiments of the present invention, the CMOS sensor ofimaging system 10 can be another suitable sensor having thecharacteristics described herein.

In certain embodiments, when heaters are not uniform, only a smallportion of melting zone 132 may be used as the ROI (the thermallyuniform portion). Typically, the uniform portion of melting zone 132corresponds to that part of melting zone 132 heated by calibratedheating elements, and the non-uniform, fringe portions of melting zone132 include non-calibrated heating elements. An effectively larger ROIcan be created under conditions of non-uniform heating by dividing thenon-uniform ROI portion of melting zone 132 into several smaller“sub-ROIs”, each of which is small enough to have a uniform temperature.In some embodiments, melt curves are plotted for each small sub-ROI andare then shifted to the temperature calibrated region in the ROImathematically to correlate the melt curves of the non-calibratedsub-ROIs with the melt curve of the ROI of the calibrated portion ofmelting zone 132. SNR could be improved with the larger effective ROI.Alternatively, in an embodiment where the heater is calibrated, a ROIover the uniformly heated section of the heater is used, so thetemperature of this specific ROI is calibrated and can be assumed to beaccurate. The edges of the heater usually have a different temperaturethan the calibrated ROI. The temperature difference between the regionson the heater edges and the calibrated ROI can be calculated. Forexample, when material in the calibrated ROI melts at 70° C. and thematerial at the edges melts at 80° C. (reading of the heater temperaturecalibrated using the ROI), it is known that the edge is 10° C. coolerthan the calibrated ROI. Edges of the heater will melt later but thetemperature axis can be adjusted using the temperature difference data.Thus, the heater edges could have the same temperature axis as thecalibrated ROI. The melt curves of more ROIs along the heater can beshifted to the calibrated ROI and added up to generate a melt curve withbetter SNR.

Reference fluorescence materials could be placed near the sample(without interfering with the fluorescence from the sample) so thatfluctuations from the light source can be identified and removed. Thatis, fluctuations from the expected fluorescence signal of the referencefluorescence materials can be identified so that corrections in thefluorescence signal measured from the sample material can be made. Inaddition, the reference fluorescence materials could be used to identifywhether distortion is from the light source or a heater when debuggingthe system. That is, distortions that exhibit in both the referencefluorescence materials and the sample materials are likely due toexcitation light source distortions, whereas distortions that exhibit inthe sample materials but not in reference fluorescence materials arelikely due to distortions in the heater. Such reference materials couldbe any fluorescence material that can be excited and detected by theimaging system 10. Suitable, non-limiting examples include Sytox blue,CFP, Alexa 647, Cy5, BODIPY650/665, or various quantum dots. Inaddition, the plastics in the microfluidic fixture could be used asfluorescence reference too.

In one embodiment, glue (e.g., Lens Bond Type SK-9 available fromSummers Optical of Hatfield, Pa.) used to bond the microfluidic-chip anda heat sink can be used as reference.

The reference fluorescence material can be chosen to be temperaturedependent. Heater fluctuation can be identified and removed using suchreference fluorescence material. For example, when proper materials arechosen which have temperature dependent fluorescence intensity(fluorescence intensity of most dyes is temperature dependent, such asAlexa Fluor 647, fluorescein, et al), the temperature fluctuation of theheaters can be monitored using the fluorescence of the reference dye.When heater temperature changes, the fluorescence intensity of thereference dyes will follow the temperature changes. When the referencematerial has sufficient intensity and area, the fluorescence signal fromthe reference material measurement will have a sufficient SNR, so thechange in fluorescence intensity could reflect the heater temperaturefluctuations. In certain embodiments, the reference material may beplaced close enough to the heaters for this purpose. The temperaturedependence does not need to be same as the sample under measurement.

In some embodiments, only one detector is needed to measure both thesample fluorescence and reference fluorescence.

In one embodiment, thermal melt data is recorded by the imaging system118 as a function of time (JPEGs are saved with their time stamps asfile name). Temperature is also recorded by the temperature controlsystem 120 as a function of time. This creates two data sets: an imagevs. time data set and a temperature vs. time data set. Preferably, thesetwo sets of data are synchronized accurately to give the correct melttemperature.

There are many ways to synchronize two independent data sets. In oneembodiment, with a DSLR, a signal can be sent from a PC to the remoteshooting control port of the camera to take a static image right beforethermal melt is started. For example, the static image signal can besent at the same time the initial heater signal is sent. Thus, both theimages and the heaters will have the same start time. The time thestatic image is taken has a known relation with both the PC time(temperature time) and camera live view image time stamps, so thetemperature and images can be synchronized, in some cases to within <30ms resolution.

When imaging a uniform object, the images usually are not ideallyuniform. The center will have higher intensity then the edges. In orderto make melt data better, the microfluidic chip can be shifted so thatmelting zone 132 (where thermal melt occurs) is as close to the centerof image as possible.

Aspects of the invention are implemented via control and computinghardware components, user-created software, data input components, anddata output components. Hardware components include computing andcontrol modules, such as main controller 130, such as microprocessorsand computers, configured to effect computational and/or control stepsby receiving one or more input values, executing one or more algorithmsstored on non-transitory machine-readable media (e.g., software) thatprovide instruction for manipulating or otherwise acting on the inputvalues, and output one or more output values. Such outputs may bedisplayed, for example, on display device 132, or otherwise indicated toa user for providing information to the user, for example, informationas to the status of the instrument or a process being performed thereby,or such outputs may comprise inputs to other processes and/or controlalgorithms. Data input components comprise elements by which data isinput for use by the control and computing hardware components. Suchdata inputs may comprise positions sensors and motor encoders, as wellas manual input elements via user input devices 134, such as key boards,touch screens, microphones, switches, manually-operated scanners, etc.Data output components may comprise hard drives or other storage media,monitors, printers, indicator lights, or audible signal elements (e.g.,buzzer, horn, bell, etc). Software comprises instructions stored onnon-transitory computer-readable media which, when executed by thecontrol and computing hardware, cause the control and computing hardwareto perform one or more automated or semi-automated processes.

Embodiments of the present invention have been fully described abovewith reference to the drawing figures. While the present invention hasbeen described and shown in considerable detail with reference tocertain illustrative embodiments, including various combinations andsub-combinations of features, those skilled in the art will readilyappreciate other embodiments and variations and modifications thereof asencompassed within the scope of the present invention. Moreover, thedescriptions of such embodiments, combinations, and sub-combinations arenot intended to convey that the inventions require features orcombinations of features other than those expressly recited in theclaims. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe following appended claims.

1. An imaging system configured to generate images of a reaction withina microchannel of a microfluidic device, the imaging system comprising:a sensor element configured to generate a storable image of at least aportion of a microchannel; and a plurality of illumination elementsdisposed with respect to the sensor element and configured to illuminatea portion of the microfluidic chip to be imaged by the sensor element,wherein at least one of the illumination elements comprises anillumination assembly comprising: an LED; a mask disposed in front ofthe LED and having an opening formed therein so as to control an areailluminated by the illumination assembly; a filter along an optic pathof the illumination assembly for controlling the spectral content oflight emitted by the illumination assembly, and a lens for imaging anarea with light emitted by the illumination assembly, wherein the LED,the mask, the filter, and the lens are aligned along an optic axis ofthe illumination assembly.
 2. The imaging system of claim 1, wherein atleast two of the illumination elements are configured to illuminatedifferent portions of the microfluidic chip.
 3. The imaging system ofclaim 1, wherein the sensor element comprises a digital single lensreflex camera.
 4. The imaging system of claim 1, wherein each of theillumination elements comprises an LED.
 5. The imaging system of claim1, comprising four illumination elements disposed at 90-degree angularincrements about the sensor element.
 6. The imaging system of claim 1,wherein the sensor element comprises a CMOS sensor.
 7. The imagingsystem of claim 6, wherein the CMOS sensor has a pixel array of up to5616×3744 pixels or larger.
 8. The imaging system of claim 1, whereinsaid sensor element includes a pixel array, and wherein said systemfurther comprises logic elements configured to detect an image of only aportion of the pixels of the pixel array.
 9. The imaging system of claim1, wherein said sensor element is configured to generate multipleimages.
 10. The imaging system of claim 1, wherein said sensor elementis configured to generate an image having a JPEG format.
 11. The imagingsystem of claim 1, further comprising reference fluorescence materialpositioned to be imaged by the sensor element along with at least aportion of a microchannel.
 12. The imaging system of claim 1, furthercomprising at least one extension tube between the sensor and themicrochannel.
 13. The imaging system of claim 1, further comprising anemission filter positioned between the sensor and the microchannel andconfigured to allow light signals of only a selected wavelength to reachthe sensor.
 14. The imaging system of claim 5, wherein the microchannelcomprises a first zone and a second zone, and wherein a first one of theLEDs is positioned and oriented to illuminate the second zone; a secondand a third of the LEDs are spaced 180-degrees from each other and aredisposed on opposed sides of the sensor and are positioned and orientedto illuminate the first zone; and a fourth one of the LEDs is positionedand oriented to illuminate both the first zone and the second zone. 15.The imaging system of claim 1, wherein each of the plurality ofillumination elements is configured to illuminate a portion of themicrofluidic chip at a prescribed wavelength.
 16. A system forperforming a nucleic acid diagnostic assay on a sample materialcomprising: microfluidic means including micro-channels for transportingsample material and for enabling an assay process to be performed onsample material within one or more portions of said micro-channels, theassay process including at least one of PCR amplification and a thermalmelt analysis; means in operative cooperation with said microfluidicmeans for introducing sample material into said micro-channels of saidmicrofluidic means; means in operative cooperation with saidmicrofluidic means for moving sample material through each micro-channelof said microfluidic means; thermal means for heating and/or cooling oneor more portions of said microfluidic means to one or more selectedtemperatures; and imaging means for imaging sample material within oneor more portions of each said micro-channel, including means for storingdata related to images created by said imaging means.
 17. The system ofclaim 16, further comprising processing means for processing datarelated to images created by said imaging means and for generating datarelating to results of at least one of said PCR amplification and saidthermal melt analysis.
 18. The system of claim 17, further comprisingcontrol means for controlling operation of said means for introducingsample material, said means for moving sample material, said thermalmeans, said imaging means, and said processing means.
 19. The system ofclaim 16, wherein said imaging means is configured for detectingfluorescent emissions of prescribed wavelengths from sample materialwithin said micro-channels and comprises: means for directing anexcitation signal of a prescribed excitation wavelength at a portion ofsaid micro-channel; and means for capturing an image of fluorescentemission of a prescribed emission wavelength from the sample materialwithin the portion of said micro-channel.
 20. A computer-implementedmethod for analyzing thermal melt data from sample material within amicrochannel, comprising: recording thermal melt image data as afunction of time with an imaging system by saving JPEG images generatedby the imaging system with time stamps; recording temperature data as afunction of time with a temperature control system; and synchronizingthe thermal melt data with the temperature data by: sending a staticrecord signal to the imaging system to cause the imaging system torecord static image data before thermal melt is started and recordingthe time of the static image data; and synchronizing the temperaturedata to the time of the static image data.
 21. The method of claim 20,comprising sending the static image signal to the imaging system at thesame time an initial heater signal is sent to the temperature controlsystem, so that the thermal melt image data and heaters controlled bythe temperature control system will have the same start time, and thensynchronizing the temperature data and the thermal melt image data basedon a relationship of the time of the static image data to the timestamps of the JPEG images of the thermal melt image data and recordedtime of the temperature data.
 22. The method of claim 20, whereinrecording thermal image data comprises: illuminating at least a portionof the microchannel; generating, with a pixel array sensor, an image offluorescence emitted by material within the illuminated portion of themicrochannel, wherein each pixel of the image has a JPEG value; defininga region of interest (“ROI”) comprising a portion of the pixels of thepixel array; and calculating the intensity of fluorescence emitted bymaterial within the microchannel by averaging the JPEG values of all thepixels in the ROI.
 23. The method of claim 22, wherein each pixel hastwo sub-pixels of a first color and one sub-pixel of a second color, andthe JPEG value of each pixel is computed as (2×first colorsub-pixel+1×second color sub-pixel)/3.
 24. The method of claim 22,further comprising dividing at least a portion of the ROI into one ormore sub-ROIs and calculating fluorescence intensity within each sub-ROIby averaging JPEG values of all the pixels in the sub-ROI.
 25. Themethod of claim 22, wherein the generating step is performed with a CMOSsensor.
 26. The method of claim 22, wherein the CMOS sensor comprises adigital single lens reflex camera.
 27. The method of claim 22, furthercomprising repeating the generating and calculating steps one or moretimes over a period of time and monitoring changes in the calculatedfluorescence intensity over a period of time.
 28. The method of claim22, comprising monitoring a fluid flow within the microchannel by:generating images of fluorescence emitted by material within theilluminated portion of the microchannel at two different times;detecting displacement of a feature of the image within from one imageto the next; and computing a time lapse from one image to the next. 29.The method of claim 22, further comprising: illuminating a referencefluorescence material positioned adjacent the microchannel; generating,with the pixel array sensor, an image of fluorescence emitted by thereference fluorescence material; and defining at least two ROIs, whereinone ROI encompasses the portion of the microchannel and a second ROIencompasses a portion of the reference fluorescence material.
 30. Themethod of claim 29, further comprising: calculating the intensity offluorescence emitted by the reference fluorescence material by averagingthe JPEG values of all the pixels in the ROI encompassing the referencefluorescence material; and adjusting the intensity of fluorescenceemitted by material within the microchannel based on the intensity offluorescence emitted by the reference fluorescence material.
 31. Theimaging system of claim 9, wherein said sensor element is configured togenerate multiple images at a frequency of up to 30 Hz.